S. aureus is capable of withstanding nearly every facet of host innate immunity, including the production of nitric oxide (NO.) NO.-resistance is not apparent in other bacteria, including closely related coagulase negative staphylococci (CNS). We have found that the key to S. aureus NO.-resistance is the ability of this organism to evoke a metabolic state, centered around lactate metabolism, that is essentially immune to NO. S. aureus harbors three lactate dehydrogenases, one of which (Ldh1) is not present in CNS and plays a critical role in S. aureus NO.-resistance. The focus of this proposal is to understand the role of lactate-metabolism in S. aureus virulence and immune evasion. Specific Aim 1. Establish Lqo as part of the NOX-cycle, an essential pathway for S. aureus virulence. We identified a gene encoding the S. aureus lacate:quinone oxidoreductase (Lqo) and demonstrated its role in virulence. Here we test hypotheses explaining why a fourth lactate-utilizing enzyme in S. aureus is so critical to pathogenesis. We will establish the NOX cycle, a metabolic pathway relying on both Ldh1 and Lqo that actually allows S. aureus to use host NO. for energy production. We will also structurally and biochemically characterize Lqo, the founder of a new family of enzymes found only among the staphylococci. Specific Aim 2. Unravel the multiple roles for lactate metabolism in S. aureus pathogenesis. Unlike CNS, S. aureus exhibits lactate enantiomer preference during NO.-stress and produces copious amounts of the L-isomer, the only form of lactate able to participate in the NOX cycle. We will define the regulatory mechanisms resulting in peak expression of ldh1, the S. aureus-specific lactate dehydrogenase responsible for enantiomer preference. We will also test hypotheses aimed at explaining the curious observation that both L- and D-lactate production are required for S. aureus virulence. We have observed that genes involved in S. aureus lactate metabolism are either not present or are differentially regulated in CNS. Thus, the new acquisition and altered regulation of genes involved in S. aureus lactate-metabolism provides a model for the Metabolic Evolution of an emerging pathogen. These incremental evolutionary changes provided S. aureus with the metabolic flexibility necessary to thrive within an immunocompetent host and must have coincided with the acquisition of canonical virulence factors for this pathogen to emerge.